2-nitroimidazole derivative, production thereof, and radiosensitizer containing the same as active ingredient

ABSTRACT

The present invention relates to a 2-nitroimidazole derivative represented by the following general formula (I): ##STR1## wherein R 1 , R 2  and R 3  may be the same or different from each other and mean individually a hydrogen atom or an acyl group, and a radiosensitizer containing the same. The compound (I) according to this invention has an excellent radiosensitizing effect and high safety, so that a radiosensitizer containing the same is remarkably useful in the radiotherapy of cancers.

TECHNICAL FIELD

This invention relates to a 2-nitroimidazole derivative, which is anovel substance useful as a radiosensitizer for hypoxic cells, aproduction process thereof, and a radiosensitizer containing the same asan active ingredient.

BACKGROUND ART

Cancers are difficult to cure even at the present day, and hence inflictgreat pains and economical losses on the world. Broadly speaking, acancer is treated by three therapies of radiotherapy, chemotherapy andsurgical therapy.

In the radiotherapy for the cancer among these, the presence of hypoxiccells in the tumor becomes the greatest problem. The hypoxic cells havehigh resistance to radioactive rays and are hence believed to be aserious cause of the refractoriness and/or the recurrence in theradiotherapy. On the other hand, no hypoxic cells exist in any normaltissues. Upon performing the radiotherapy for the cancer, it istherefore important to enhance the radiosensitivity of the hypoxic cellsin the tumor.

In view of the foregoing circumstances, the present inventors carriedout an extensive investigation with a view toward providing a medicamentby which the radiosensitivity of normal cells is unchanged and onlyhypoxic cells are sensitized when radiotherapy is performed, i.e., aradiosensitizer for hypoxic cells (hereinafter referred to as"radiosensitizer"). As a result, it has been found that a2-nitroimidazole derivative represented by the general formula (I) whichwill be described subsequently has a high radiosensitizing effect evenin a low concentration and is also low in toxicity which has heretoforebecome the greatest problem, leading to completion of the presentinvention.

DISCLOSURE OF THE INVENTION

The present invention provides a 2-nitroimidazole derivative representedby the following general formula (I): ##STR2## wherein R₁, R₂ and R₃ maybe the same or different from each other and mean individually ahydrogen atom or an acyl group, and a production process thereof. Thepresent invention further provides a radiosensitizer containing thisderivative as an active ingredient.

BEST MODE FOR CARRYING OUT THE INVENTION

In the general formula (I) by which the compound according to thisinvention is represented, examples of the acyl groups indicated by R₁,R₂ and R₃ include alkanoyl, alkenoyl or aroyl groups having 1-10 carbonatoms. The illustrative representatives of the compounds (1) accordingto this invention are as follows:

(1)1-[(2',3'-diacetoxy-1'-acetoxymethyl)propoxy]methyl-2-nitroimidazole;

(2)1-[(2',3'-dipropionoxy-1'-propionoxymethyl)propoxy]methyl-2-nitroimidazole;

(3)1-[(2',3'-diacryloxy-1'-acryloxymethyl)propoxy]methyl-2-nitroimidazole;

(4)1-[(2',3'-dicrotonoxy-1'-crotonoxymethyl)propoxy]methyl-2-nitroimidazole;

(5)1-[(2',3'-dibenzoxy-1'-benzoxymethyl)propoxy]methyl-2-nitroimidazole;and

(6)1-[(2',3'-dihydroxy-1'-hydroxymethyl)propoxy]methyl-2-nitroimidazole.

The compound (I) of this invention can be produced in accordance with,for example, the following reaction formula: ##STR3## wherein R₁ ', R₂ 'and R₃ ' may be the same or different from each other and meanindividually an acyl group, R₄ denotes an acyl group, and X stands for ahydrogen atom or a trialkylsilyl group.

Namely, 1,2,4-triacyloxy-3-acyloxymethoxybutane (II) is reacted with2-nitroimidazole or a trialkylsilyl-containing product thereof (III),thereby producing a compound (Ia) in which R₁, R₂ and R₃ in the formula(I) are individually an acyl group. Further, the hydrolysis of thecompound (Ia) produces a compound (Ib) in which R₁, R₂ and R₃ in theformula (I) are individually a hydrogen atom.

In the above-described reaction formula, the reaction in the case wherethe compound (III) is 2-nitroimidazole is preferably carried out bymelting the compound (II) and 2-nitroimidazole in the presence of acatalyst while reducing pressure. Various kinds of catalysts may be usedas such a catalyst. For example, proton acids such asparatoluenesulfonic acid, methanesulfonic acid and trichloroacetic acid,and Lewis acids such as anhydrous zinc chloride, anhydrous aluminumchloride and anhydrous stannic chloride may be used. Among these,paratoluenesulfonic acid is the most effective catalyst owing toformation of little by-product. The proportions of the compound (II) and2-nitroimidazole to be used may be optionally determined. However, it isgenerally desirable that the former compound should be used in a rangeof from an amount equimolar to the latter compound to an amount slightlyin excess of the same. In general, the reaction is preferably conductedat a temperature of 50°-150° C. The reaction time may vary according tothe reaction reagents, solvent, temperature, reaction-acceleratingsubstance, etc. to be used, but is generally 30 minutes to 6 hours.

On the other hand, the reaction in the case where the compound (III) is1-trialkylsilyl-2-nitroimidazole is preferably carried out by reactingthe compound (II) with 1-trialkylsilyl-2-nitroimidazole in the presenceof a Lewis acid.

1-Trialkylsilyl-2-nitroimidazole which is a raw compound can be obtainedas a raw material usable as it is by reacting 2-nitroimidazole withN,O-bistrialkylsilylacetamide in an excess amount while stirring them atroom temperature or under heat and then distilling under reducedpressure the unreacted silylating agent out of the reaction mixture.

Various kinds of acids may be used as the Lewis acid. As exemplary Lewisacids, may be mentioned anhydrous stannic chloride, anhydrous aluminumchloride, anhydrous zinc chloride and the like. These acids maypreferably be used in a catalytic amount or an amount equivalent to thecompound (II). The proportions of the compound (II) and1-trialkylsilyl-2-nitroimidazole to be used may be optionallydetermined. However, it is generally preferred that the compound (II)should be used in a range of from an amount equimolar to1-trialkylsilyl-2-nitroimidazole to an amount slightly in excess of thesame. Various kinds of solvents may be used as a solvent for thereaction. For example, acetonitrile, methylene chloride, benzene,toluene and the like are however preferred. The reaction may beperformed at a temperature ranging from -30° to 50° C. However, it isgenerally preferred to conduct the reaction under water cooling or atroom temperature. The reaction time may vary according to the reactionreagents, temperature, reaction solvent, catalyst, etc. to be used.However, it is generally preferred to conduct the reaction for 30minutes to 6 hours.

After completion of the reaction according to any one of theabove-described two processes, the intended product is separated fromthe reaction mixture and purified by a method known per se in the art.For example, the reaction mixture is subjected to extraction, and theextract is washed with water, concentrated and then purified bypreparative thin-layer chromatography, column chromatography or thelike, thereby obtaining a compound (Ia) with high yield.

The hydrolysis reaction of the compound (Ia) is conducted by a method inwhich the compound (Ia) is treated overnight under ice cooling or atroom temperature in an absolute alcohol containing sodium alcoholatetherein or an absolute alcohol with ammonia gas saturated therein, amethod in which the compound (Ia) is hydrolyzed with an organic basesuch as triethylamine or pyridine in a water-containing alcohol at atemperature ranging from room temperature to 80° C., or the like.Examples of the alcohol used include lower alcohols such as methylalcohol, ethyl alcohol and propyl alcohol.

The compound (I) of this invention is low in toxicity as demonstrated byTest Example which will be described subsequently, and has an excellentradiosensitizing effect. The compound (I) of this invention ispreferably administered 15 minutes through 5 hours prior to theirradiation of radioactive rays. The administration is carried outorally or parenterally. The compound (I) may be prepared in the form oftablets, capsules, granules, powder, suppository or injection bysuitably adding additives such as an excipient, stabilizer, preservativeand buffer. The dose the compound (I) may vary according to age, thesite of tumorigenesis, the kind of tumor, symptoms and the like.However, it is generally preferable to dose the compound (I) in anamount of 0.2 g through 5.0 g/m² of area of body surface.

The acute toxicity test and radiosensitizing effect of the compound (I)according to this invention will hereinafter be described by thefollowing test examples.

(1) Acute toxicity test

Using ICR male mice after the elapse of 5 weeks from their birth,1-[(2',3'-dihydroxy-1'-hydroxy)propoxy]methyl-2-nitroimidazole [therepresentative compound (6)] dissolved in physiological saline wasintravenously administered to them. The mice were observed over 14 daysafter the administration to determine the 50% lethal dose (LD_(50/14)).As a result, it was found to be 5,900 mg/kg.

(2) Test of radiosensitizing effect 1) In vitro test 1

Cell used: Single cells of BMT-6

Radioactive ray irradiated: ⁶⁰ Co - γ rays

Oxygen-reducing treatment: A mixed gas composed of 95% nitrogen and 5%carbon dioxide gas is caused to flow into a cell suspension.

Judgment of survival rate of the cells: According to a colony countingmethod ##EQU1##

The result obtained under the above-described conditions is as follows:

ER of the representative compound (6) in a concentration of 1 mM: 1.67

2) In vivo test

Animal used: Balb/c mice

Tumor used: BMT-6

Substance tested: Representative compound (6) 200 mg/kg

Dosing manner: Intravenous administration of the representative compound(6) dissolved in physiological saline 15 minutes prior to theirradiation of radioactive rays

Radioactive ray irradiated: ⁶⁰ Co - γ rays, the whole body irradiation

Judgment of radiosensitizing effect: A radiosensitization ratio (ER) wasdetermined from the dose and the reduction percent of tumor cells toconduct the judgment of the effect.

The result obtained under the above-described conditions is as follows:

ER of the representative compound (6) in a dose of 200 mg/kg: 1.44

EXAMPLES

The present invention will hereinafter be described in more detail bythe following Reference Example and Examples. However, it should beborne in mind that this invention is not limited to and by theseexamples.

REFERENCE EXAMPLE 1 Synthesis of1,3,4-triacetoxy-2-acetoxymethoxy-butane

(1) Fifty grams of meso-erythritol were dispersed in 700 ml of pyridine.To the resulting dispersion, 3 equivalent weights (125.3 g) of aceticanhydride were added dropwise under water cooling so as to graduallyform a transparent solution. Most of pyridine was then distilled out ofthe solution by a rotary evaporator. The residue was added with 500 mlof ethyl acetate, thereby subjecting it to extraction. The extract wasthen washed with water. Thereafter, the ethyl acetate layer was dried byanhydrous sodium sulfate and then concentrated. The concentrate wassubjected to column chromatography on silica gel. First of all, elutionwas carried out with benzene, thereby obtainingtetraacetoxy-meso-erythritol. Then, 26 g of1,3,4-triacetoxy-meso-erythritol was eluted with a 9:1 mixed solvent ofbenzene and ethyl acetate.

(2) To 26 g of 1,3,4-triacetoxy-meso-erythritol thus obtained, 200 ml ofdimethoxyethane was added to mix them into a solution. Phosphoruspentoxide was added to the resultant solution while stirring it underwater cooling. At this time, the addition was conducted while checkingby TLC (developing solution: benzene:ethyl acetate=1:1). The addition ofphosphorus pentoxide was stopped at the time when a TLC became one spot,thereby completing the reaction. A supernatant liquid of the reactionmixture was shifted to a separatory funnel, thereby subjecting it toextraction with 200 ml of n-hexane. The extract was then added with anaqueous saturated solution of sodium bicarbonate. The resulting mixturewas washed with water and dried by anhydrous sodium sulfate. The solventwas then distilled out of the mixture, thereby obtaining 30.5 g of1,3,4-triacetoxy-2-methoxymethoxy-butane.

(3) To 30.5 g of 1,3,4-triacetoxy-2-methoxymethoxybutane obtained in thestep (2), 15 g of acetic anhydride was added to mix them. The resultantmixture was cooled with ice. After thoroughly cooled with ice, 4 ml ofboron trifluoride etherate was added dropwise to react them for 1 hourwith stirring. After completion of the reaction, the reaction mixturewas poured into iced water mixed with a great excess of sodiumbicarbonate, thereby decomposing unreacted acetic anhydride toneutralize the reaction mixture. Thereafter, 200 ml of ethyl acetate wasadded, thereby subjecting the reaction mixture to extraction. Theextract was washed with water and dried (by sodium sulfate), therebyobtaining 30.0 g of 1,3,4-triacetoxy-2-acetoxymethoxy-butane.

EXAMPLE 1 Synthesis of1-[(2',3'-diacetoxy-1'-acetoxymethyl)propoxy]methyl-2-nitroimidazole

A flask was charged with 5.6 g of 2-nitroimidazole, 16.0 g of1,3,4-triacetoxy-2-acetoxymethoxybutane and 0.5 g of p-toluenesulfonicacid monohydrate. A trap was attached to the flask to connect it toaspirator so as to permit the reduction of the pressure within theflask. The flask was immersed in an oil bath of 130°-140° C. whilereducing the pressure within the flask and stirring the contents. Aceticacid distilled off as a reaction advanced. The reaction was completed inabout 15 minutes. After the reaction mixture was cooled to roomtemperature, about 300 ml of ethyl acetate was added to the reactionmixture, thereby subjecting it to extraction. The extract was washedwith an aqueous saturated solution of sodium hydrogencarbonate and thenwith water. The thus-washed extract was dried by anhydrous sodiumsulfate and then concentrated under reduced pressure. The residue waspurified by preparative high speed liquid chromatography on a silica gelcolumn making use of a mixed solvent of ethyl acetate-benzene as aneluting solution, thereby obtaining the title compound (15.3 g, yield:52%) as a viscous oil.

MS (m/e): 373 (M⁺)

IR (cm⁻¹): 1740(CO), 1535(NO₂), 1490(NO₂)

NMR (δ, CDCl₃) 2.0(s,9H,CH₃ COx3), 3.6-4.4(m,5H,--CH₂ OAcx2, ═CH--),5.1(m,1H,═CH--OAc), 7.1 (s,1H,ring proton), 7.3(s,1H,ring proton)

EXAMPLE 2 Synthesis of1-[(2',3'-dihydroxy-1'-hydroxymethyl)propoxy]methyl-2-nitroimidazole

In 50 ml of absolute methanol, 3.73 g of1-[(2',3'-diacetoxy-1'-acetoxymethyl)propoxy]methyl-2-nitroimidazole wasdissolved. While stirring the resulting solution at room temperature, a5% solution of sodium ethoxide in absolute ethanol was added dropwiseuntil the pH of the solution turned to 9.0. After stirring the resultingreaction mixture for 3 hours at room temperature, "Dowex 50W" (H⁺,product of Dow Chemical Co.) was slowly added until the pH of thereaction mixture turned to 7.0. After Dowex 50W was then removed byfiltration under reduced pressure, the solvent was distilled off underreduced pressure, and the residue was recrystallized from ethanol,thereby obtaining 1.48 g (yield: 60%) of the title compound as paleyellow needle crystals.

MS (m/e): 248 (M⁺)

IR (cm⁻¹): 3450(OH), 1540(NO₂), 1490(NO₂)

NMR [δ, DMSO(d₆)]: 3.2-3.6(m,7H,═CH--OH,--CH₂ OHx2, ═CH--),4.1-4.6(m,3H,OHx3), 5.8(s,2H,--OCH₂ N═), 7.1 (s,1H,ring proton),7.7(s,1H,ring proton)

EXAMPLE 3 Synthesis of1-[(2,3-diacetoxy-1-acetoxymethyl)propoxy]methyl-2-nitroimidazole

A flask equipped with a calcium chloride cylinder was charged with 5.6 gof 2-nitroimidazole and 30 ml of N,O-bistrimethylsilylacetamide (BSA).The contents were stirred at 40°-50° C. to gradually form a transparentsolution. One through two hours later, 2-nitroimidazole was completelyconverted into a silyl-containing compound. Unreacted BSA andO-trimethylsilylacetamide as a by-product were distilled off underreduced pressure at a temperature of 90° C. or higher to use them in thenext reaction as they are. The thus-obtained silyl-containing2-nitroimidazole was added with 16.0 g of1,3,4-triacetoxy-2-acetoxymethoxybutane and 20 ml of anhydrousacetonitrile. Then, 10 ml of anhydrous stannic chloride was addeddropwise under water cooling. The contents were stirred for 2-3 hours toreact them. The reaction mixture was poured into 500 ml of ethyl acetatecontaining 100 g of ice therein. The mixture was stirred while furtheradding anhydrous sodium hydrogencarbonate until bubbles of carbondioxide became free from generation. After the ethyl acetate layer waswashed with water and dried by anhydrous sodium sulfate, the solvent wasdistilled off under reduced pressure. The residue was purified bypreparative high speed liquid chromatography on a silica gel columnmaking use of a mixed solvent of ethyl acetatebenzene as an elutingsolution, thereby obtaining the title compound (15.1 g, yield: 81%).

EXAMPLE 4 Synthesis of1-[(2',3'-dihydroxy-1'-hydroxymethyl)propoxy]methyl-2-nitroimidazole

In 100 ml of methanol containing 10% of water, 3.73 g of1-[(2',3'-diacetoxy-1'-acetoxymethyl)propoxy]methyl-2-nitroimidazole wasdissolved. To the resulting solution, 10 g of triethylamine was added toreact them for 4 hours at room temperature with stirring. Aftercompletion of the reaction, the solvent was distilled off by anevaporator, and the residue was recrystallized from ethanol, therebyobtaining the title compound as colorless needle crystals with a yieldof 57%.

INDUSTRIAL APPLICABILITY

The compound (I) according to this invention has an excellentradiosensitizing effect and high safety, so that a radiosensitizercontaining the compound (I) is remarkably useful in the radiotherapy ofcancers.

We claim:
 1. A 2-nitroimidazole derivative represented by the followinggeneral formula (I): ##STR4## wherein R₁, R₂ and R₃ may be the same ordifferent from each other and mean individually a hydrogen atom or anacyl group.
 2. A radiosensitizer containing, as an active ingredient,the 2-nitroimidazole derivative as set forth in claim 1 and an additive.